Monodisperse Picoliter Droplets for Low-Bias and Contamination-Free Reactions in Single-Cell Whole Genome Amplification

Whole genome amplification (WGA) is essential for obtaining genome sequences from single bacterial cells because the quantity of template DNA contained in a single cell is very low. Multiple displacement amplification (MDA), using Phi29 DNA polymerase and random primers, is the most widely used method for single-cell WGA. However, single-cell MDA usually results in uneven genome coverage because of amplification bias, background amplification of contaminating DNA, and formation of chimeras by linking of non-contiguous chromosomal regions. Here, we present a novel MDA method, termed droplet MDA, that minimizes amplification bias and amplification of contaminants by using picoliter-sized droplets for compartmentalized WGA reactions. Extracted DNA fragments from a lysed cell in MDA mixture are divided into 10⁵ droplets (67 pL) within minutes via flow through simple microfluidic channels. Compartmentalized genome fragments can be individually amplified in these droplets without the risk of encounter with reagent-borne or environmental contaminants. Following quality assessment of WGA products from single Escherichia coli cells, we showed that droplet MDA minimized unexpected amplification and improved the percentage of genome recovery from 59% to 89%. Our results demonstrate that microfluidic-generated droplets show potential as an efficient tool for effective amplification of low-input DNA for single-cell genomics and greatly reduce the cost and labor investment required for determination of nearly complete genome sequences of uncultured bacteria from environmental samples.     

A Simple Method for Encapsulating Single Cells in Alginate Microspheres Allows for Direct PCR and Whole Genome Amplification

Microdroplets are an effective platform for segregating individual cells and amplifying DNA. However, a key challenge is to recover the contents of individual droplets for downstream analysis. This paper offers a method for embedding cells in alginate microspheres and performing multiple serial operations on the isolated cells. Rhodobacter sphaeroides cells were diluted in alginate polymer and sprayed into microdroplets using a fingertip aerosol sprayer. The encapsulated cells were lysed and subjected either to conventional PCR, or whole genome amplification using either multiple displacement amplification (MDA) or a two-step PCR protocol. Microscopic examination after PCR showed that the lumen of the occupied microspheres contained fluorescently stained DNA product, but multiple displacement amplification with phi29 produced only a small number of polymerase colonies. The 2-step WGA protocol was successful in generating fluorescent material, and quantitative PCR from DNA extracted from aliquots of microspheres suggested that the copy number inside the microspheres was amplified up to 3 orders of magnitude. Microspheres containing fluorescent material were sorted by a dilution series and screened with a fluorescent plate reader to identify single microspheres. The DNA was extracted from individual isolates, re-amplified with full-length sequencing adapters, and then a single isolate was sequenced using the Illumina MiSeq platform. After filtering the reads, the only sequences that collectively matched a genome in the NCBI nucleotide database belonged to R. sphaeroides. This demonstrated that sequencing-ready DNA could be generated from the contents of a single microsphere without culturing. However, the 2-step WGA strategy showed limitations in terms of low genome coverage and an uneven frequency distribution of reads across the genome. This paper offers a simple method for embedding cells in alginate microspheres and performing PCR on isolated cells in common bulk reactions, although further work must be done to improve the amplification coverage of single genomes.     

Quantitative evaluation by minisequencing and microarrays reveals accurate multiplexed SNP genotyping of whole genome amplified DNA

Whole genome amplification (WGA) procedures such as primer extension preamplification (PEP) or multiple displacement amplification (MDA) have the potential to provide an unlimited source of DNA for large-scale genetic studies. We have performed a quantitative evaluation of PEP and MDA for genotyping single nucleotide polymorphisms (SNPs) using multiplex, four-color fluorescent minisequencing in a microarray format. Forty-five SNPs were genotyped and the WGA methods were evaluated with respect to genotyping success, signal-to-noise ratios, power of genotype discrimination, yield and imbalanced amplification of alleles in the MDA product. Both PEP and MDA products provided genotyping results with a high concordance to genomic DNA. For PEP products the power of genotype discrimination was lower than for MDA due to a 2-fold lower signal-to-noise ratio. MDA products were indistinguishable from genomic DNA in all aspects studied. To obtain faithful representation of the SNP alleles at least 0.3 ng DNA should be used per MDA reaction. We conclude that the use of WGA, and MDA in particular, is a highly promising procedure for producing DNA in sufficient amounts even for genome wide SNP mapping studies.     

Improved multiple displacement amplification with phi29 DNA polymerase for genotyping of single human cells

The ability to genotype multiple loci of single cells would be of significant benefit to investigations of cellular processes such as oncogenesis, meiosis, fertilization, and embryogenesis. We report a simple two-step, single-tube protocol for whole-genome amplification (WGA) from single human cells using components of the GenomiPhi V2 DNA Amplification kit. For the first time, we demonstrate reliable generation of 4-7 microg amplified DNA from a single human cell within 4 h with a minimum amount of artifactual DNA synthesis. DNA amplified from single cells was genotyped for 13 heterozygous short tandem repeats (STRs) and 7 heterozygous single nucleotide polymorphisms (SNPs), and the genotyping results were compared with purified genomic DNA. Accuracy of genotyping (percent of single-cell amplifications genotyped accurately for any particular STR or SNP) varied from 37% to 100% (with an average of 80%) for STRs and from 89% to 100% (averaging 94%) for SNPs. We suggest that the method described in this report is suitable for WGA from single cells, the product of which can be subsequently used for many applications, such as preimplantation genetic analysis (PGD).     

Genome amplification of single sperm using multiple displacement amplification

Sperm typing is an effective way to study recombination rate on a fine scale in regions of interest. There are two strategies for the amplification of single meiotic recombinants: repulsion-phase allele-specific PCR and whole genome amplification (WGA). The former can selectively amplify single recombinant molecules from a batch of sperm but is not scalable for high-throughput operation. Currently, primer extension pre-amplification is the only method used in WGA of single sperm, whereas it has limited capacity to produce high-coverage products enough for the analysis of local recombination rate in multiple large regions. Here, we applied for the first time a recently developed WGA method, multiple displacement amplification (MDA), to amplify single sperm DNA, and demonstrated its great potential for producing high-yield and high-coverage products. In a 50 μl reaction, 76 or 93% of loci can be amplified at least 2500- or 250-fold, respectively, from single sperm DNA, and second-round MDA can further offer >200-fold amplification. The MDA products are usable for a variety of genetic applications, including sequencing and microsatellite marker and single nucleotide polymorphism (SNP) analysis. The use of MDA in single sperm amplification may open a new era for studies on local recombination rates.     

An isothermal primer extension method for whole genome amplification of fresh and degraded DNA: applications in comparative genomic hybridization, genotyping and mutation screening

We describe a protocol that uses a bioinformatically optimized primer in an isothermal whole genome amplification (WGA) reaction. Overnight incubation at 37 degrees C efficiently generates several hundred- to several thousand-fold increases in input DNA. The amplified product retains reasonably faithful quantitative representation of unamplified whole genomic DNA (gDNA). We provide protocols for applying this isothermal primer extension WGA protocol in three different techniques of genomic analysis: comparative genomic hybridization (CGH), genotyping at simple tandem repeat (STR) loci and screening for single base mutations in a common monogenic disorder, beta-thalassemia. gDNA extracted from formalin-fixed paraffin-embedded (FFPE) tissues can also be amplified with this protocol.     

Microarray analysis of copy number variation in single cells

We present a protocol for reliably detecting DNA copy number aberrations in a single human cell. Multiple displacement-amplified DNAs of a cell are hybridized to a 3,000-bacterial artificial chromosome (BAC) array and to an Affymetrix 250,000 (250K)-SNP array. Subsequent copy number calling is based on the integration of BAC probe-specific copy number probabilities that are estimated by comparing probe intensities with a single-cell whole-genome amplification (WGA) reference model for diploid chromosomes, as well as SNP copy number and loss-of-heterozygosity states estimated by hidden Markov models (HMM). All methods for detecting DNA copy number aberrations in single human cells have difficulty in confidently discriminating WGA artifacts from true genetic variants. Furthermore, some methods lack thorough validation for segmental DNA imbalance detection. Our protocol minimizes false-positive variant calling and enables uniparental isodisomy detection in single cells. Additionally, it provides quality assessment, allowing the exclusion of uninterpretable single-cell WGA samples. The protocol takes 5-7 d.     

Exploring whole genome amplification as a DNA recovery tool for molecular genetic studies.

The whole genome amplification (WGA) protocol evaluated during this study, GenomiPhi DNA amplification kit, is a novel method that is not based on polymerase chain reaction but rather relies on the highly processive and high fidelity Phi29 DNA polymerase to replicate linear genomic DNA by multiple strand displacement amplification. As little as 1 ng of genomic DNA template is sufficient to produce microgram quantities of high molecular weight DNA. The question explored during this study is whether such a WGA method is appropriate to reliably replenish and even recover depleted DNA samples that can be used for downstream genetic analysis. A series of human DNA samples was tested in our laboratory and validated using such analytical methods as gene-specific polymerase chain reaction, direct sequencing, microsatellite marker analysis, and single nucleotide polymorphism allelic discrimination using TaqMan and Pyrosequencing chemistries. Although degraded genomic DNA is not a good template for Phi29 WGA, this method is a powerful tool to replenish depleted DNA stocks and to increase the amount of sample for which biological tissue availability is scarce. The testing performed during the validation phase of the study indicates no discernable difference between WGA samples and the original DNA templates. Thus, GenomiPhi WGA can be used to increase precious or depleted DNA stocks, thereby extending the life of a family-based linkage analysis project and increasing statistical power.     

The Performance of Whole Genome Amplification Methods and Next-Generation Sequencing for Pre-Implantation Genetic Diagnosis of Chromosomal Abnormalities

Reliable and accurate pre-implantation genetic diagnosis(PGD) of patient’s embryos by next-generation sequencing(NGS) is dependent on efficient whole genome amplification(WGA) of a representative biopsy sample. However, the performance of the current state of the art WGA methods has not been evaluated for sequencing. Using low template DNA(15 pg) and single cells, we showed that the two PCR-based WGA systems Sure Plex and MALBAC are superior to the REPLI-g WGA multiple displacement amplification(MDA) system in terms of consistent and reproducible genome coverage and sequence bias across the 24 chromosomes, allowing better normalization of test to reference sequencing data. When copy number variation sequencing(CNV-Seq) was applied to single cell WGA products derived by either Sure Plex or MALBAC amplification, we showed that known disease CNVs in the range of 3e15 Mb could be reliably and accurately detected at the correct genomic positions. These findings indicate that our CNV-Seq pipeline incorporating either Sure Plex or MALBAC as the key initial WGA step is a powerful methodology for clinical PGD to identify euploid embryos in a patient’s cohort for uterine transplantation.     

Evaluation of multiple displacement amplification in a 5 cM STR genome-wide scan

Multiple displacement amplification (MDA) has emerged as a promising new method of whole genome amplification (WGA) with the potential to generate virtually unlimited genome-equivalent DNA from only a small amount of seed DNA. To date, genome-wide high marker density assessments of MDA–DNA have focussed mainly upon suitability for single nucleotide polymorphism (SNP) genotyping applications. Suitability for short tandem repeat (STR) genotyping has not been investigated in great detail, despite their inherent instability during DNA replication, and the obvious challenge that this presents to WGA techniques. Here, we aimed to assess the applicability of MDA in STR genotyping by conducting a genome-wide scan of 768 STR markers for MDAs of 15 high quality genomic DNAs. We found that MDA genotyping call and accuracy rates were only marginally lower than for genomic DNA. Pooling of three replicate MDAs resulted in a small increase in both call rate and genotyping accuracy. We identified 34 STRs (4.4% of total markers) of which five essentially failed with MDA samples, and 29 of which showed elevated genotyping failures/discrepancies in the MDAs. We emphasise the importance of DNA and MDA quality checks, and the use of appropriate controls to identify problematic STR markers.     

Whole-genome amplification-based GenomiPhi for multiple genomic analysis of individual early porcine embryos

The multiple displacement amplification (MDA) method, which relies on isothermal DNA amplification using the DNA polymerase of the bacteriophage phi29, was recently developed for high-performance, whole-genome amplification (WGA). The objective of the present study was to determine whether a target sequence could be successfully amplified by conventional PCR when the genomic DNA of a single Day-7 porcine blastocyst (derived from SCNT of a gene-engineered fibroblast) was amplified by the MDA method and used as a template. The yield of double-stranded DNA was 103.5 ± 16.0 ng/embryo (range, 75-125), as assessed by a PocoGreen assay. However, non-specific products (20 ± 5 ng/tube) were also generated, even in the negative control. Thus, ∼81% of the 103.5 ng (84 ng) of amplified DNA was estimated to be porcine sequences (2.2 × 10³-fold enrichment). In addition, PCR confirmed the presence of transgenes, as well as endogenous α-1,3-galactosyltransferase and homeobox Nanog genes in all embryos. Sequencing of the amplified products verified the fidelity of this system. In conclusion, the MDA-mediated WGA, which was simple, inexpensive, and did not require a thermal cycler, could be a powerful tool for multiple genomic analyses of individual early porcine embryos.     

Concurrent Whole-Genome Haplotyping and Copy-Number Profiling of Single Cells

Methods for haplotyping and DNA copy-number typing of single cells are paramount for studying genomic heterogeneity and enabling genetic diagnosis. Before analyzing the DNA of a single cell by microarray or next-generation sequencing, a whole-genome amplification (WGA) process is required, but it substantially distorts the frequency and composition of the cell’s alleles. As a consequence, haplotyping methods suffer from error-prone discrete SNP genotypes (AA, AB, BB) and DNA copy-number profiling remains difficult because true DNA copy-number aberrations have to be discriminated from WGA artifacts. Here, we developed a single-cell genome analysis method that reconstructs genome-wide haplotype architectures as well as the copy-number and segregational origin of those haplotypes by employing phased parental genotypes and deciphering WGA-distorted SNP B-allele fractions via a process we coin haplarithmisis. We demonstrate that the method can be applied as a generic method for preimplantation genetic diagnosis on single cells biopsied from human embryos, enabling diagnosis of disease alleles genome wide as well as numerical and structural chromosomal anomalies. Moreover, meiotic segregation errors can be distinguished from mitotic ones.     

不同单细胞全基因组扩增方法的比较及MALBAC在辅助生殖中的应用

单细胞全基因组扩增(whole genome amplification, WGA)是指在单细胞水平对全基因组进行扩增的新技术,其原理是将分离的单个细胞的微量全基因组DNA进行扩增,获得高覆盖率的完整的基因组后进行高通量测序,用于揭示细胞异质性。目前,WGA方法主要包括引物延伸预扩增(primer extension preamplification PCR, PEP-PCR)、简并寡核苷酸引物PCR (degenerate oligonucleotide primed PCR, DOP-PCR)、多重置换扩增(multiple displacement amplification, MDA)、多次退火环状循环扩增(multiple annealing and looping-based amplification cycles, MALBAC)等。本文对不同的单细胞WGA方法的原理及应用情况分别进行了阐述,并对其扩增效率进行评价和比较,包括基因组覆盖度、均一性、重现性、SNV (single-nucleotide variants)和CNV (copy number variants)检测力等。综合对比不同单细胞WGA方法后发现,MALBAC的扩增均一性最高、等位基因脱扣率最低、重现性最好,且对于CNV和SNV的检测效果最好。本文还阐述了MALBAC技术在人类单精子减数重组、非整倍体分析以及人类卵细胞基因组研究中的应用。     

Single-nucleotide-polymorphism genotyping for whole-genome-amplified samples using automated fluorescence correlation spectroscopy

Whole-genome amplification (WGA) methods were adopted for single-nucleotide-polymorphism (SNP) typing to minimize the amount of genomic DNA that has to be used in typing for thousands of different SNPs in large-scale studies; 5-10 ng of genomic DNA was amplified by a WGA method (improved primer-extension-preamplification-polymerase chain reaction (I-PEP-PCR), degenerated oligonucleotide primer-PCR (DOP-PCR), or multiple displacement amplification (MDA)). Using 1/100 to 1/500 amounts of the whole-genome-amplified products as templates, subsequent analyses were successfully performed. SNPs were genotyped by the sequence-specific primer (SSP)-PCR method followed by fluorescence correlation spectroscopy (FCS). The typing results were evaluated for four different SNPs on tumor necrosis factor receptor 1 and 2 genes (TNFR1 and TNFR2). The genotypes determined by the SSP-FCS method using the WGA products were 100% in concordance with those determined by nucleotide sequencing using genomic DNAs. We have already carried out typing of more than 300 different SNPs and are currently performing 7,500-10,000 typings per day using WGA samples from patients with several common diseases. WGA coupled with FCS allows specific and high-throughput genotyping of thousands of samples for thousands of different SNPs.     

Detailed chromosomal and molecular genetic analysis of single cells by whole genome amplification and comparative genomic hybridisation.

Molecular genetic analysis of isolated single cells and other minute DNA samples is limited because there is insufficient DNA to perform more than one independent PCR amplification. One solution to this problem is to first amplify the entire genome, thus providing enough DNA for numerous subsequent PCRs. In this study we have investigated four different methods of whole genome amplification performed on single cells, and have identified a protocol that generates sufficient quantities of DNA for comparative genomic hybridisation (CGH) as well as more than 90 independent amplification reactions. Thus, numerous specific loci and the copy number of every chromosome can be assessed in a single cell. We report here the first reliable application of CGH to single cells from human preimplantation embryos (blastomeres) and to single fibroblasts, buccal cells and amniocytes.     

Efficient de novo assembly of single-cell bacterial genomes from short-read data sets

Whole genome amplification by the multiple displacement amplification (MDA) method allows sequencing of DNA from single cells of bacteria that cannot be cultured. Assembling a genome is challenging, however, because MDA generates highly nonuniform coverage of the genome. Here we describe an algorithm tailored for short-read data from single cells that improves assembly through the use of a progressively increasing coverage cutoff. Assembly of reads from single Escherichia coli and Staphylococcus aureus cells captures >91% of genes within contigs, approaching the 95% captured from an assembly based on many E. coli cells. We apply this method to assemble a genome from a single cell of an uncultivated SAR324 clade of Deltaproteobacteria, a cosmopolitan bacterial lineage in the global ocean. Metabolic reconstruction suggests that SAR324 is aerobic, motile and chemotaxic. Our approach enables acquisition of genome assemblies for individual uncultivated bacteria using only short reads, providing cell-specific genetic information absent from metagenomic studies.     

Simple Bulk Readout of Digital Nucleic Acid Quantification Assays

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are still not routinely applied, due to the cost and specific equipment associated with commercially available methods. Here we present a simplified method for readout of digital droplet assays using a conventional real-time PCR instrument to measure bulk fluorescence of droplet-based digital assays.We characterize the performance of the bulk readout assay using synthetic droplet mixtures and a droplet digital multiple displacement amplification (MDA) assay. Quantitative MDA particularly benefits from a digital reaction format, but our new method applies to any digital assay. For established digital assay protocols such as digital PCR, this method serves to speed up and simplify assay readout.Our bulk readout methodology brings the advantages of partitioned assays without the need for specialized readout instrumentation. The principal limitations of the bulk readout methodology are reduced dynamic range compared with droplet-counting platforms and the need for a standard sample, although the requirements for this standard are less demanding than for a conventional real-time experiment. Quantitative whole genome amplification (WGA) is used to test for contaminants in WGA reactions and is the most sensitive way to detect the presence of DNA fragments with unknown sequences, giving the method great promise in diverse application areas including pharmaceutical quality control and astrobiology.     

Whole genome amplification of Chelex-extracted DNA from a single mite: a method for studying genetics of the predatory mite Phytoseiulus persimilis

We developed and optimized a method using Chelex DNA extraction followed by whole genome amplification (WGA) to overcome problems conducting molecular genetic studies due to the limited amount of DNA obtainable from individual small organisms such as predatory mites. The DNA from a single mite, Phytoseiulus persimilis Athias-Henrot (Acari: Phytoseiidae), isolated in Chelex suspension was subjected to WGA. More than 1000-fold amplification of the DNA was achieved using as little as 0.03 ng genomic DNA template. The DNA obtained by the WGA was used for polymerase chain reaction followed by direct sequencing. From WGA DNA, nuclear DNA intergenic spacers ITS1 and ITS2 and a mitochondrial DNA 12S marker were tested in three different geographical populations of the predatory mite: California, the Netherlands, and Sicily. We found a total of four different alleles of the 12S in the Sicilian population, but no polymorphism was identified in the ITS marker. The combination of Chelex DNA extraction and WGA is thus shown to be a simple and robust technique for examining molecular markers for multiple loci by using individual mites. We conclude that the methods, Chelex extraction of DNA followed by WGA, provide a large quantity of DNA template that can be used for multiple PCR reactions useful for genetic studies requiring the genotypes of individual mites.     

Assessment of multiple displacement amplification in molecular epidemiology

Well-characterized epidemiological resources are generated with great effort, yet associated patient DNA samples can be limiting. The efficacy of the whole genome amplification (WGA) method, termed multiple displacement amplification (MDA), was assessed for detecting heterozygous sequence variants, mutation scanning, and PCR for challenging segments. Fifteen common polymorphisms from 10 genes located on 8 chromosomes were genotyped by direct sequencing of 300 PCR products from 115 high-quality MDA-amplified DNA samples extracted by different methods. The GC content of these analyzed segments ranges from 30% to 69%. Genotyping results demonstrate 100% accuracy. For heterozygotes, the relative intensity of peaks generated by the two alleles is highly similar for genomic and MDA-amplified genomic DNA, independent of GC content. In contrast, one of four heterozygous loci was mistyped when lower quality MDA-amplified DNA samples were used. The results of single-stranded conformation polymorphism (SSCP)-type of mutation scanningfor seven MDA-amplified DNA samples in four genes were concordant with the genomic DNA samples. PCR on MDA-amplified DNA was routinely successful for challenging 10- and 12-kb segments with GC content ranging from 30% to 80%, demonstrating that rather long segments, which are difficult to amplify with PCR, are amplified well with MDA. These results suggest that MDA is an effective method of WGA with utility in molecular epidemiology. Quality control of the MDA-amplified DNA is critical for high performance.     

Effects of DNA mass on multiple displacement whole genome amplification and genotyping performance

Background

Whole genome amplification (WGA) promises to eliminate practical molecular genetic analysis limitations associated with genomic DNA (gDNA) quantity. We evaluated the performance of multiple displacement amplification (MDA) WGA using gDNA extracted from lymphoblastoid cell lines (N = 27) with a range of starting gDNA input of 1–200 ng into the WGA reaction. Yield and composition analysis of whole genome amplified DNA (wgaDNA) was performed using three DNA quantification methods (OD, PicoGreen^® and RT-PCR). Two panels of N = 15 STR (using the AmpFlSTR^® Identifiler^® panel) and N = 49 SNP (TaqMan^®) genotyping assays were performed on each gDNA and wgaDNA sample in duplicate. gDNA and wgaDNA masses of 1, 4 and 20 ng were used in the SNP assays to evaluate the effects of DNA mass on SNP genotyping assay performance. A total of N = 6,880 STR and N = 56,448 SNP genotype attempts provided adequate power to detect differences in STR and SNP genotyping performance between gDNA and wgaDNA, and among wgaDNA produced from a range of gDNA templates inputs.

Results

The proportion of double-stranded wgaDNA and human-specific PCR amplifiable wgaDNA increased with increased gDNA input into the WGA reaction. Increased amounts of gDNA input into the WGA reaction improved wgaDNA genotyping performance. Genotype completion or genotype concordance rates of wgaDNA produced from all gDNA input levels were observed to be reduced compared to gDNA, although the reduction was not always statistically significant. Reduced wgaDNA genotyping performance was primarily due to the increased variance of allelic amplification, resulting in loss of heterozygosity or increased undetermined genotypes. MDA WGA produces wgaDNA from no template control samples; such samples exhibited substantial false-positive genotyping rates.

Conclusion

The amount of gDNA input into the MDA WGA reaction is a critical determinant of genotyping performance of wgaDNA. At least 10 ng of lymphoblastoid gDNA input into MDA WGA is required to obtain wgaDNA TaqMan^® SNP assay genotyping performance equivalent to that of gDNA. Over 100 ng of lymphoblastoid gDNA input into MDA WGA is required to obtain optimal STR genotyping performance using the AmpFlSTR^® Identifiler^® panel from wgaDNA equivalent to that of gDNA.